Forerunner Genes: A Novel Class of Early Detection Markers for Bladder Cancer Identification of early changes associated with the development of preneoplastic conditions would provide important clue on early events of carcinogenesis and could aid in the development of novel markers for early identification of occult neoplasia. We performed genome-wide search for losses of genetic material on multiple DNA samples corresponding to normal urothelium, in situ preneoplastic lesions, and invasive carcinoma extracted from the entire mucosal surface of resected human bladders. The analysis of hits associated with growth advantage of preneoplastic lesions allowed us to identify six chromosomal regions mapping to 3q22.1,5q22.2-5q23.3,9q22.12, 10q26.1, 13q14,and 17p13 that may be critical for the development of bladder cancer and contain a distinct class of genes referred to as forerunner (FR) genes involved in the clonal expansion of precursor conditions. We concentrated our efforts on one of these regions, which contain a model tumor suppressor RB1. We used high-resolution whole-organ mapping with SNPs, which facilitated the identification of three positional candidate FR genes (ITM2B, P2RY5and CHC1L) mapping contiguously to RB1. We also provide evidence that their inactivation is likely to be critical for the development of preneoplastic lesions. Using combined genetic and epigenetic mapping of the same region we have identified an additional FR gene contiguous to RB1 (GPR38). The expression, mutational, and methylation analyses showed that at least two of the prototypic FR genes (ITM2B and GPR38) are frequently methylated in bladder cancer and their methylation can be detected in voided urine samples. This proposal describes our continuing studies focused on the role and function of the prototypic FR gene (ITM2B) contiguous to RB1. Our preliminary data implicate that this gene plays a key role in controlling proliferation of preneoplastic clone and is frequently methylated in bladder cancer. Therefore, in addition to the functional studies we propose to use the frequently methylated FR genes combined with other methylation markers for the detection of bladder cancer. The specific aims for our study are as follows: Specific Aim 1. Clarify the role and function of the FR gene (ITM2B) in bladder cancer. Specific Aim 2. Identify an effective methylation panel of genes, including FR genes for detecting bladder cancer in urine. Specific Aim 3. Validate the methylation panel of biomarkers in a prospective cohort of patients at high risk of bladder cancer recurrence.